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Fluopsin C is an antibiotic compound derived from secondary metabolism of different microorganisms, which possesses antitumor, antibacterial, and antifungal activity. Related to fluopsin C antiproliferative activity, the aim of this study was to examine the following parameters: cytotoxicity, genotoxicity, cell cycle arrest, cell death induction (apoptosis), mitochondrial membrane potential (MMP), colony formation, and mRNA expression of genes involved in adaptive stress responses and cellular death utilizing a monolayer. In addition, a three-dimensional cell culture was used to evaluate the effects on growth of tumor spheroids. Fluopsin C was cytotoxic (1) producing cell division arrest in the G1 phase, (2) elevating expression of mRNA of the CDKN1A gene and (3) decrease in expression of mRNA H2AFX gene. Further, fluopsin C enhanced DNA damage as evidenced by increased expression of mRNA of GADD45A and GPX1 genes, indicating that reactive oxygen species (ROS) may be involved in the observed genotoxic response. Reticulum stress was also detected as noted from activation of the ribonuclease inositol-requiring protein 1 (IRE1) pathway, since a rise in mRNA expression of the ERN1 and TRAF2 genes was observed. During the cell death process, an increase in mRNA expression of the BBC3 gene was noted, indicating participation of this antibiotic in oncotic (ischemic) cell death. Data thus demonstrated for the first time that fluopsin C interferes with the volume of tumor spheroids, in order to attenuate their growth. Our findings show that fluopsin C modulates essential molecular processes in response to stress and cell death.

This study aimed to evaluate the modes of action of harringtonine (HT) and homoharringtonine (HHT) alkaloids in cell with wild (HepG2/C3A) and mutant p53 (HuH-7.5). We performed assays for cytotoxicity, genotoxicity, induction of apoptosis, cell cycle phase, and membrane integrity. Obtained data were compared with the relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. The relative expression of the genes showed an increase in apoptosis-inducing mRNAs, such as TNF and BBC3, as well as a reduction in BCL2 and BAK. The mRNAs of CYP2E1 and CYP2C19 xenobiotic metabolism genes increased in both lineages, while CYP3A4 increased only in the HuH-7.5 lineage. The mRNA expression of endoplasmic reticulum (ER) stress genes ( ERN1 and EIF2AK3) was shown to increase in HHT and HT treatments. A similar increase was recorded in the mRNA expression of the TRAF2 gene. The changes observed in this study support the hypothesis that ER stress was more strongly associated with TNF induction, causing cell death by apoptosis in p53 mutant cells. This result with wild and mutant p53 cells may have clinical implications in the use of these compounds.

β-Glucans (βG) are polysaccharides widely distributed in nature with chemopreventive properties. The aim of this study was to investigate the effects of βG and the combined treatment with doxorubicin (Dox) on cell viability and mRNA levels of genes involved in cell cycle, apoptosis and antioxidant response. βG was not cytotoxic. The mRNA levels of CCNA2 of cells exposed to β-glucan was upregulated and the exposure to Dox decreased the expression, while the combination led to an upregulation. Modulation of mRNA levels of CASP9 suggest that βG could inhibit promotion and progression steps of carcinogenesis, eliminating neoplastic cells. The upregulation of CCNA2 gene in combined treatment could be occurred due to ability of βG in restoring the cell cycle distribution pattern after treatment with Dox. The upregulation of SOD1 suggests that βG can enhance the intracellular antioxidant defense, reducing the levels of superoxide dismutase induced by Dox. This response could reduce oxidative damage and attenuate tissue damage during chemotherapeutic treatment. Our data suggest that the drug combination may be less effective in killing tumor cells than the treatment with Dox alone. Thus, future studies should carefully consider this effect on indication of βG during chemotherapy β-Glucans (βG) are polysaccharides widely distributed in nature with chemopreventive properties. The aim of this study was to investigate the effects of βG and the combined treatment with doxorubicin (Dox) on cell viability and mRNA levels of genes involved in cell cycle, apoptosis and antioxidant response. βG was not cytotoxic. The mRNA levels of CCNA2 of cells exposed to β-glucan was upregulated and the exposure to Dox decreased the expression, while the combination led to an upregulation. Modulation of mRNA levels of CASP9 suggest that βG could inhibit promotion and progression steps of carcinogenesis, eliminating neoplastic cells. The upregulation of CCNA2 gene in combined treatment could be occurred due to ability of βG in restoring the cell cycle distribution pattern after treatment with Dox. The upregulation of SOD1 suggests that βG can enhance the intracellular antioxidant defense, reducing the levels of superoxide dismutase induced by Dox. This response could reduce oxidative damage and attenuate tissue damage during chemotherapeutic treatment. Our data suggest that the drug combination may be less effective in killing tumor cells than the treatment with Dox alone. Thus, future studies should carefully consider this effect on indication of βG during chemotherapy

Made available in DSpace on 2021-06-25T11:43:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01. Added 1 bitstream(s) on 2021-07-15T15:05:47Z : No. of bitstreams: 1 S1516-89132020000100331.pdf: 533608 bytes, checksum: 8f247c86cd1b6e458914c584126394d0 (MD5) Fundacao de Apoio ao Desenvolvimento do Ensino, Ciencia e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The alpha-tomatine is a steroidal glycoalkaloid found in immature tomatoes (Lycopersicon esculentum) that has important biological functions including the inhibition of cancer cell growth and preventing metastasis. This study aimed to evaluate the effects of alpha-tomatine on cytotoxicity, cellular proliferation, apoptosis, and mRNA expression of APC, CCNA2, beta-catenin, CASP9, BAK, BAX and BCL-XL in colorectal adenocarcinoma cell line HT-29. HT29 cells were treated with three concentrations of alpha-tomatine (0.1, 1 and 10 mu g/mL), although only the 1 mu g/mL concentration of alpha-tomatine was used to evaluate genetic expression patterns by real time-PCR. Results showed that alpha-tomatine was cytotoxic only at the 10 mu g/mL concentration. Cell proliferation was significantly inhibited after the first 24 hours of treatment only with concentrations of 10 mu g/mL. In contrast, there were no significant differences in apoptosis for any treatment. In the gene expression studies, only APC expression was significantly altered by alpha-tomatine treatment. In conclusion, tomatine has antiproliferative activity in the first 24h of treatment, does not induce apoptosis in this cell line and causes disruption of cell membranes, thereby increasing the expression of APC gene related to cell cycle. Univ Estadual Paulista, Rio Claro, SP, Brazil Univ Fed Mato Grosso do Sul, Fac Med, Campo Grande, MS, Brazil Univ Estadual Londrina, Londrina, Parana, Brazil Univ Estadual Paulista, Rio Claro, SP, Brazil

Background: Genistein (5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is the most abundant isoflavone in soybean, which has been associated with a lower risk of development of cancer and cardiovascular diseases. Of particular interest regarding cancer preventive properties of flavo-noids is their interaction with cytochrome P450 enzymes (CYPs). However, contradictory data report the effect of genistein on expression of СYPs enzymes. Objective: The aim of this study was to investigate the effects of genistein on cytochrome P450 (CYP) gene expression levels in human hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cells. Methods: Real-time RT–PCR was used to examine the expression of genes families involved in xenobi-otic metabolism, such as CYP1 (CYP1A1, CYP1B1), CYP2 (CYP2E1, CYP2D6), CYP3 (CYP3A4); and of a family involved in the catabolism of the all-trans-retinoic acid (ATRA), CYP26 (CYP26A1, CYP26B1). Results: RT-qPCR data analysis showed that after 12 h of exposure of HepG2/C3A cells to genistein (5 and 50 µM) there was an upregulation of CYP1A1 and CYP1B1 and downregulation of CYP2D6, CYP26A1 and CYP26B1 mRNA levels. There was no change in the mRNA levels of CYP P450 genes in HT29 cells. Conclusion: Our results suggest that treatment with genistein in non-toxic concentrations may impact the expression level of CYPs involved in the biotransformation of xenobiotics and drug metabolizing en-zymes. Moreover, the downregulation of ATRA metabolism-related genes opens a new research path for the study of genistein as retinoic acid metabolism blocking agent for treating cancer and other patholo-gies.

Studies that evaluated the mechanisms of action of Plumbagin (PLB) and its toxicity may contribute to future therapeutic applications of this compound. We investigate biomarker important in the mechanisms of action correlate the expression of mRNA with the cytotoxic and genotoxic effects of PLB on HepG2/C3A. In the analysis of cytotoxicity, PLB decreased cell viability and membrane integrity at concentrations ≥ 15μM. Xenobiotic-metabolizing system showed strong mRNA induction of CYP1A1, CYP1A2, and CYP3A4, suggesting extensive metabolization. PLB induced apoptosis and an increase in the mRNA expression of genes BBC3, CASP3, and CASP8. At a concentration of 15μM, there was a reduction in the expression of PARP1 mRNA and an increase in the expression of BECN1 mRNA, suggesting that PLB may also induce cell death by autophagy. PLB induced an arrest at the G2/M phase due to DNA damage, as observed in the comet assay. This damage is associated with the increased mRNA expression of genes p21, GADD45A, and H2AFX and with changes in the expression of proteins H2AX, p21, p53, Chk1, and Chk2. These results allow a better understanding of the cellular action of PLB and of its toxicity, thereby contributing to the development of PLB-based drugs, with markers of mRNA expression possibly playing a role as indicators for monitoring toxicity in human cells.

Plumbagin (PLB) is a phytochemical being used for centuries in traditional medicines. Recently, its capacity to inhibit the development of human tumors has been observed, through the induction of apoptosis, cell cycle arrest, and inhibition of angiogenesis and metastasis. Here we evaluated the mechanism of action of PLB in the kidney adenocarcinoma 786-O cell line, which are metabolizing cells important for toxicology studies. After the treatment with PLB, we observed increased apoptosis and cell cycle arrest in S and G2/M phases, starting at 5 µM. In addition, PLB was cytotoxic, genotoxic and induced loss of cell membrane integrity. Regarding gene expression, treatment with 7.5 µM PLB reduced the amount of MTOR, BCL2 and ATM transcripts, and increased CDKN1A (p21) transcripts. Phosphorylation levels of yH2AX was increased and MDM2 protein level was reduced following the treatment with PLB, demonstrating its genotoxic effect. Our results suggest that PLB acts in molecular pathways related to the control of proliferation and cell death in 786-O cells.

Soybean isoflavonoids have received significant attention due to their potential anticarcinogenic and antiproliferative effects and possible role in many signal transduction pathways. However, their mechanisms of action and their molecular targets remain to be further elucidated. In this paper, we demonstrated that two soybean isoflavones (genistein and daidzein) reduced the proliferation of the human colon adenocarcinoma grade II cell line (HT-29) at concentrations of 25 and 50-100 μM, respectively. We then investigated the effects of genistein and daidzein by RT-PCR on molecules that involved in tumor development and progression by their regulation of cell proliferation. At a concentration of 50 μM genistein, there was suppressed expression of β-catenin (CTNNBIP1). Neither genistein nor daidzein affected APC (adenomatous polyposis coli) or survivin (BIRC5) expression when cells were treated with concentrations of 10 or 50 μM. These data suggest that the down-regulation of β-catenin by genistein may constitute an important determinant of the suppression of HT-29 cell growth and may be exploited for the prevention and treatment of colon cancer.

The installation of oxidative process arises from an imbalance between oxidants and antioxidants compounds, in favor of excessive generation of free radicals. This process can affect cellular components, like endoplasmic reticulum (ER). The ER is extremely sensitive to changes in homeostasis, where, on different stimuli, may result in adaptation to survival or induction of apoptosis (unfolded protein response, “UPR” pathway). The oxidative damage caused by endogenous or exogenous agents or a dysregulation of the UPR pathway can lead to adverse conditions, whose chronicity has important implications for the etiology of chronic diseases, including cancer. Therefore, it becomes important to search for chemoprotective agents aiming their role in preventive medicine. Between these substances, selenium’s antioxidant activity seems to be effective in treating diseases which have the oxidative stress as its development. We evaluated the modulating action of l-(+)-selenomethionine (SeMet) in HepG2 cells against cellular stress induced by H2O2 through MTT assay, comet assay, and gene expression by qRT-PCR of genes related to oxidative stress, UPR pathway, and apoptosis. In MTT assay, the lower concentrations of SeMet showed a cytoprotective action against the damage caused by H2O2. Likewise, it was verified in the comet assay that the concentration of 50 ng/mL reduced the genotoxic damage caused by H2O2. SeMet at 50 ng/mL regulated the genes tested in the qRT-PCR, showing an antiapoptotic and antioxidant effect. These results suggest that SeMet positively modulates the genes of oxidative stress and ER, leading to a chemoprotective and antioxidant effect, becoming an alternative in preventive medicine.

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity.